The present invention relates generally to the fields of microbiology, pharmacology, and medicine. More specifically, the present invention relates to allosteric therapeutics for microbial infections and associated glucosylating toxins.
Clostridium difficile (C. difficile) infection (CDI) is the most prevalent cause of hospital-acquired infectious diarrhea and life-threatening colitis worldwide (Kelly et al., N Engl J Med 359:1932-1940 (2008)). Two large exotoxins, TcdA (308 kDa) and TcdB (270 kDa), are secreted from the majority of C. difficile bacterial strains that cause disease in humans, and there is little ambiguity that these toxins are pathogenic since toxin-deficient strains are avirulent (Savidge et al., Gastroenterology 125:413-20 (2003); Lyras et al. Nature 458:1176-79 (2009); Kuehne et al., Nature 467:711-13 (2010)). The clostridial glucosylating toxins and the multifunctional autoprocessing repeats-in-toxins (MARTX) share a common virulence mechanism for cell entry that represents a potential target for therapeutic intervention (Fullner et al., Infect Immun 75: 5079-84 (2007); Sheahan et al., EMBO J 26: 2552-61 (2007); Egerer et al., J Biol Chem 282: 25314-21 (2007); Pei et al., Protein Science 18: 856-62 (2009)). Cellular internalization of these exotoxins is dependent on cytosolic inositol hexakisphosphate (InsP6) allosteric cofactor, which activates an autocatalytic cysteine protease domain to facilitate toxin self-cleavage (Reineke et al., Nature 446:415-19 (2007); Prochazkova et al., J Biol Chem 283: 23656-64 (2008); Lupardus et al., Science 322:265-68 (2008); Egerer et al., J Biol Chem 284:3389-95 (2009)). Intracellular release of the smaller N-terminus glucosyltransferase effector domain results in the mono-O glucosylation of small GTPases of the Rho family, including RhoA, Rad, and Cdc42 (Popoff et al., Biochim Biophys Acta. 88:797-812 (2009)). Glucosylation of Rho proteins inhibits their molecular switch function, thus blocking Rho GTPase-dependent signaling in intestinal epithelial cells, leading to alterations in the actin cytoskeleton, fluid secretion, acute inflammation, and necrosis of the colonic mucosa.
Host defense mechanisms that might be employed to protect against the clostridial glucosylating toxins are not well defined, although C. difficile toxins are potent inducers of nitric oxide (NO), which is known to be protective against these toxins (Ng et al., Gastroenterology Apr. 13 (2010)). However, the precise molecular mechanism for the protective effects of NO remains unknown. Some diverse signaling cascades associated with NO production are attributed to S-nitrosothiol (SNO) species that act via covalent modification of specific cysteine residues in target molecules (S-nitrosylation)(Hess, et al., Nature Rev 6:150-166 (2005)) and aberrant S-nitrosylation may play a role in disease-etiology (M W et al., Trends Mol Med 15: 391-404 (2009)).
There is a recognized need in the art for alternative therapies for infections by toxins such as Clostridium difficile. Specifically, the prior art is deficient in treatments for glucosylating toxin infections. The present invention fulfills this long-standing need and desire in the art.